The work carried out this year may be divided into four separate projects. Of these, two represent a continuation of studies begun earlier on the presence and identification of intranuclear glycoproteins. Considerable progress has been made in identifying the nuclear HMG proteins as being responsible for the aberrant hydrazide labeling reaction and points to the probability of a role for specific nuclear protein crosslinking. Further evidence has also been obtained to confirm the presence and identification of intranuclear glycogen, a project which has taken on added significance in the light of recent reports documenting an unsuspected role for glycogen as an essential requirement for nuclei formation around naked DNA in an in vitro system. A new project undertaken this year has resulted in the development of a specific assay for polysialic acid, a critical component of neural cell adhesion molecules (N-CAMs). Finally, a series of genetic studies have been initiated in an attempt to isolate the gene encoding MHL-1 as a prelude to knock-out experiments designed to provide insight into the unique role of the hepatic asialoglycoprotein receptor. GRANT-Z01DK17002 As part of a program in which the enzymes that participate in the process of detoxication are being examined as to their catalytic mechanism, attention is being concentrated on a sulfotransferase (aryl sulfotransferase IV). The enzyme catalyzes the transfer of the sulfuryl group from 3-phosphoadenosine 5-phosphosulfate (PAPS) to a very broad range of phenols; the products are 3-phosphoadenosine 5-phosphate (PAP) and the corresponding phenol sulfate. Thereby, lipophilic and toxic phenols are converted to more water soluble compounds that are readily excreted. One of the characteristics of enzymes that participate in detoxication is just this large variety of lipophilic xenobiotics that serve as substrates. This laboratory has cloned the sulfotransferase from rat liver mRNA and can express it in very large quantities from Escherichia coli. In purifying the recombinant enzyme, however, two distinct forms of the sulfotransferase were physically separable. Labeled as forms A and B, both catalyzed the reverse reaction, i.e. the formation of free phenol from the phenol sulfate and PAP, but only form B could catalyze the physiological reaction. Further investigation revealed that form A had PAP bound to it so tightly that it would not allow the physiological donor, PAPS, to bind to the enzyme. The question under investigation is the nature of the tight binding of PAP to form A which presently appears to result from protein refolding after migration of a disulfide bond.